Meiosis is a specialized cell division that generates spores or gametes that are half the genome equivalent of the progenitor cell. Homologous chromosomes segregate in meiosis I, whereas sister chromatids segregate in meiosis II. Improper segregation of homologous chromosomes in meiosis I results in aneuploid gametes. Aneuploidy is the leading cause of spontaneous abortions, as well as developmental abnormalities in viable offspring (e.g., Down syndrome). Accurate segregation of homologous chromosomes depends on a process known as recombination. Meiotic recombination is initiated by the formation of DNA double-strand breaks (DSBs) catalyzed by the evolutionarily conserved Spo11 protein. In budding yeast, at least nine other proteins are required for DSB formation, among them Rec104, but their precise roles in DSB formation are not clear. Previous studies in our laboratory have shown that Rec104 is phosphorylated during meiosis. Rec104 phosphorylation is independent of DSB formation, but depends on other DSB proteins, leading to the hypothesis that Rec104 phosphorylation is important for its role in meiosis. The long-term objective of the research described in this proposal is to elucidate the molecular mechanism and regulation of meiotic DSB formation. Specifically, studies will seek to characterize the functional role of Rec104 phosphorylation, and to uncover other meiotic roles of Rec104. The first specific aim is to identify in vivo Rec104 phosphorylation sites and determine their functional relevance. Phosphorylation sites will be mapped, and the meiotic phenotype of a non-phosphorylatable rec104 mutant will be evaluated, testing the hypothesis that Rec104 phosphorylation is important for its role in meiosis, specifically in DSB formation. The second specific aim is to identify the kinase(s) responsible for Rec104 phosphorylation. Kinases that are likely candidates for phosphorylating Rec104 will be screened by examining the Rec104 protein mobility shift in the absence of kinase activity, followed by in vitro kinase assays. The third specific aim is to uncover post-DSB roles of Rec104. Two approaches will be taken: a genetic screen for separation-of-function rec104 alleles, and Rec104 protein depletion in meiotic cultures after DSB formation.